Nontypeable Haemophilus influenza (NTHi) are Gram-negative bacilli that represent a common cause of otitis media in children. The pathogenesis of otitis media caused by NTHi is not clear. It is believed that these organisms initiate infection by colonization of epithelial cells in the upper respiratory tract and middle ear. We previously immortalized a chinchilla middle ear epithelial cell line (CMEC) and a rat middle ear epithelial cell line (RMEC). In order to study the colonization process with the two newly established cell lines, the adherence of 46 NTHi to CMEC and RMEC as well as to human conjunctiva epithelial cells (HCEC, CCL-20.2), was examined by viable count method and microscopy. All 46 strains were classified into four types: Type 1 consisted of 35 strains that adhere to all three cell lines; Type 2 consisted of four strains that adhere to human and chinchilla epithelial cells; Type 3 consisted of four strains that adhere to human and rat epithelial cells; and Type 4 consisted of three strains that adhere to none of the three cell lines. Forty-three of 46 strains among type 1 to 3 adhered to human epithelial cells more avidly than to chinchilla or rat epithelial cells. The adherence of NTHi to human "O" erythrocytes was studied by hemagglutination assay in which 34% (12/35) of type 1 strains showed positive reaction. The expression of high molecular weight proteins (HMW) and fimbrin among these NTHi strains was further studied by Western blot analysis. Thirty-four strains from types 1 to 3 expressed 120 and/or 125 Kd HMW, and 41 strains expressed the fimbrin. These data indicated that NTHi can adhere to CMEC, RMEC, and HCEC, and the expression of HMW and fimbrin among NTHi strains appears to correlate with the NTHi adherence. These epithelial cell lines are useful for cellular and molecular pathogenesis studies of otitis media.